In Vivo Imaging of Endogenously Produced HClO in Zebrafish and

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In Vivo Imaging of Endogenously Produced HClO in Zebrafish and Mice Using a Bright, Photostable Ratiometric Fluorescent Probe Chong Duan, Miae Won, Peter Verwilst, Junchao Xu, Hyeong Seok Kim, Lintao Zeng, and Jong Seung Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00224 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Analytical Chemistry

In Vivo Imaging of Endogenously Produced HClO in Zebrafish and Mice Using a Bright, Photostable Ratiometric Fluorescent Probe Chong Duan,†,§ Miae Won,‡,§ Peter Verwilst, ‡,§ Junchao Xu,† Hyeong Seok Kim,‡ Lintao Zeng *,† and Jong Seung Kim *,‡ †

Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, Tianjin

University of Technology, Tianjin 300384, P.R. China. E-mail: [email protected]. ‡

Department of Chemistry, Korea University, Seoul 02841, Korea. E-mail: [email protected]

§

These authors contributed equally to this work.

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Abstract A high brightness red fluorescent probe (S-BODIPY) has been developed for the sensitive and specific imaging of HClO/ClO– in vitro and in vivo. This probe exhibits some distinctive features such as excellent resistance to photobleaching, a high fluorescence brightness, high selectivity as well as a good biocompatibility. Upon oxidation of the thio-ether group into sulfoxide, the probe showed a noticeable ratiometric fluorescence response toward ClO– with fast response (within 30 s) and a low detection limit (59 nM). The probe demonstrated the successful imaging of exogenous and endogenous HClO/ClO– in living HeLa cells, zebrafish and mice with high signal to noise ratios. S-BODIPY allows for the real-time monitoring the level of ClO– in living cells by ratiometric fluorescence imaging, opening up exciting prospects to develop red and even near-infrared BODIPYs with high brightness and good photostability for in vivo imaging.

Introduction Hypochlorous acid (HClO) is one of the most important reactive oxygen species (ROS) in living organism and plays a vital role in various biological processes.1 Endogenous HClO is produced from hydrogen peroxide and chloride anions, catalyzed by myeloperoxidase in neutrophils in vivo as a part of the innate host defence system during microbial invasion.2 In the cellular environment it mainly leads to protein oxidation, fragmentation, cross-linking and enzyme deactivation, as well as polynucleotide cross-linking and radical generation.3,4 Furthermore, the reaction with these targets also results in the generation of downstream ROS and RNS (reactive nitrogen species), most notably chloramines. Due to these deleterious effects, HClO levels are tightly associated with several diseases, such as cardiovascular disease,5 inflammatory diseases,6 acute lung injuries,7 nephropathies,8 cystic fibrosis,9 neurodegenerative disorders10 as well as cancer11. The physiologically relevant levels of HClO in living organism are 5-25 μM.12 Therefore, it is of great importance to develop an accurate and reliable method for the in situ monitoring of biological HClO in living organisms. Fluorescence imaging is a particularly suitable providing high biocompatible imaging technique, as it is non-invasive, while spatiotemporal resolution enabling real-time visualization of


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the dynamics of subcellular events.13-15 To date several selective sensors for HClO/ClO– have been reported, based on fluorescein,16 rhodamine,17-19 cyanine,20,21 boron-dipyrromethene (BODIPY) ,22 1,8-naphthalimide23 and carbazole24. Although these probes provided means for the intracellular visualization of hypochlorite in vivo, serious drawbacks such as short excitation and emission wavelengths, poor photostability, weak fluorescence, slow reactivities and elevated toxicities have prevented broad applications. Additionally, hypochlorite-sensitive fluorophores generally operate by modulating the fluorescence intensity,25,26 and as such are unable to overcome interferences caused by intracellular microenvironments as well as photo-bleaching.27 By contrast, ratiometric fluorescence enhancement, utilizing the ratio of emission intensity at two different wavelengths provide a built-in correction and thus facilitate quantification with increased reliability.28,29 Fortunately, many ratiometric fluorescent probes for the intracellular visualization of hypochlorite have been developed in recent years and most of them can overcome the above-mentioned drawbacks.30-36 But fluorescent probes with excellent properties for high-resolution imaging in multiple living animals are still very rare. Therefore, it is important to prepare a photostable and biocompatible fluorescent probes for accurate imaging of biological HClO in multiple living animals to assess the usefulness of this probe. BODIPYs are one of the privileged scaffolds in fluorophore design, due to characteristic features such as high fluorescence quantum yields, excellent photostabilities, combined with orthogonal reactivities.37,38 Moreover, its low cytotoxicity, insensitivity to the polarity and pH, and chemical robustness render it ideal for in vivo imaging.39 Herein, we report a novel BODIPY-based ratiometric fluorescent probe S-BODIPY for in situ imaging of ClO− in vitro and in vivo. S-BODIPY can overcome many of the above-mentioned drawbacks, owing to its an excellent photostability, high fluorescence quantum yield and stable fluorescence output against pH changes, combined with its low cytotoxicity and good membrane permeability. Thus, this probe represents an ideal indicator for tracking ClO− as demonstrated both in zebrafish and mice.

EXPERIMENTAL SECTION Materials and General Methods Unless otherwise stated, all reagents were purchased from Sigma-Aldrich and used without



further purification. The 1H NMR and

13C

NMR spectra were recorded on a Bruker AV-400

spectrometer. Mass spectra were measured on a HP-1100 LC-MS spectrometer. UV-vis spectra were recorded on Hitachi UV-3310 spectrometer. Fluorescence spectra were recorded on a Hitachi FL-4500 fluorometer. Confocal microscopy fluorescence images were acquired on a Nikon A1 laser-scanning confocal microscope. Fluorescence quantum yields were determined relative to Rhodamine B (Φ = 0.65 in ethanol).40 Synthesis and characterization data of probe 5F-BODIPY (330 mg, 0.80 mmol),

40

4-(methylsulfanyl) benzaldehyde (66 mg, 0.44 mmol)

and acetic acid (0.05 mL) were placed in a round bottom flask with 25 mL of toluene. The reaction mixture was heated to 110 oC in nitrogen atmosphere, and then piperidine (0.05 mL) was added. The resulting mixture was refluxed for 4 h. After the reaction was completed, the toluene was removed under reduced pressure. Flash chromatographic purification on silica gel (eluent: Hexane/EtOAc, gradient 10/0 to 7/3) afforded compound S-BODIPY as a purple solid (150.2 mg, 34.3%). 1H NMR (400 MHz,CDCl3): δ 7.64-7.60 (d, J = 16.3 Hz, 1H), 7.53-7.51 (d, J = 8.1 Hz, 2H), 7.25-7.23 (m, 3H), 6.68 (s, 1H), 6.08 (s, H), 2.61 (s, 3H). 2.52 (s, 3H). 1.67 (s, 3H). 1.63 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 157.2, 154.8, 140.9, 140.8, 137.5, 133.1, 132.8, 131.5, 128.2, 126.4, 122.3, 121.0, 118.8, 118.1, 109.8, 15.5, 15.0, 13.9, 13.7 ppm; HRMS (ESI): m/z [M+H]+ =549.1341; calcd for C27H20BF7N2S :548.1328. Cytotoxicity assay The cytotoxicity of S-BODIPY in HeLa cells was determined by using the Cell Counting Kit-8. HeLa cells were seeded in a 96-well plate with a density of 5000 cells per well (Corning). After cell attachment for 24 h, the plate was then washed with 100 μL/well PBS, and these cells were incubated with various concentrations of S-BODIPY (1.25, 2.5, 5, 7.5, 10 and 12.5 μM) for 24 h. After that, the cells were washed with serum-free DMEM once, and then 100 μL serum-free DMEM containing 10% CCK-8 was added to each well and incubated for 1 h. The absorbance was measured at 450 nm on a plate reader. Cell viability rate was calculated according to a reported literature procedure. 34


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Cell culture and fluorescence imaging HeLa cells (Perking Union Medical College, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA) and penicillin (100 units/mL)-streptomycin (100 μg/mL) liquid (Invitrogen Corp., Carlsbad, CA) at 37 °C in a humidified incubator containing 5% CO2 in air. Before imaging experiments, HeLa cells were seeded in confocal plates (2 × 104 cells/mL). After 24 h, HeLa cells were incubated with S-BODIPY (1 µM) at 37 °C for 30 min, washed twice with PBS, and then different concentrations of ClO− was added for laser-scanning confocal microscopy measurement. Fluorescence images were captured using a Nikon A1 laser-scanning confocal microscope. By mediating the yellow channel images (λex = 543 nm, λem = 552–617 nm) with the related red channel image (λex =543 nm, λem = 648–708 nm) at the same area of HeLa cells, the ratiometric fluorescence images for ClO− were determined. Fluorescence imaging of ClO− in zebrafish The zebrafishes were incubated with an E3 embryo medium containing S-BODIPY (1 µM) for 30 min at 28 °C. After washing three times with the culture medium, the S-BODIPY-loaded zebrafishes were treated with different concentrations of ClO− for imaging measurements. For imaging endogenous HClO in zebrafish, four-day old zebrafishes were cultured in E3 embryo medium containing LPS (Escherichia coli 055:B5, 10 μg/kg, Sigma) for 24 h, and then incubated with S-BODIPY (1 µM) for 30 min. The zebrafish was washed thrice with the culture medium, and mounted on microscope stage. For control, four-day old zebrafishes were cultured in E3 embryo medium containing S-BODIPY (1 µM) for 30 min, then washed thrice with the culture medium and ready for fluorescence imaging. Fluorescence images were captured using a Nikon A1 laser-scanning confocal microscope. Fluorescence imaging of acute hepatic injury in mice models Male mice (C57Bl/6, 8 weeks old) were purchased from Orientbio (Seoul, Korea) and were used for the LPS/D-GalN-induced acute hepatic injury mice model. Mice were maintained in the Korea University animal facility under specific pathogen-free conditions and received humane



care according to the Korea University Institutional Animal Care Use Committee guidelines. To induce acute liver failure (ALF), the mice were injected intraperitoneally with LPS (Escherichia coli 055:B5, 10 μg/kg, Sigma) and D-GalN (700 mg/kg, Sigma) 12 h before S-BODIPY injection. Mice were i.v. injected with a freshly prepared S-BODIPY solution (1 mM, 30% EtOH, 30% DMF). After 2 h, the fluorescence imaging of endogenous HClO were performed with an in vivo imaging system (Maestro, CRi Inc., Woburn, MA, USA) using an emission range from 550–610 nm (yellow channel) to 610–670 nm (red channel) filter.

Results and discussion Design and Synthesis of S-BODIPY To develop a red emitting ratiometric fluorescent probe for HClO/ClO− in living organisms, 5F-BODIPY underwent a Knoevenagel condensation with 4-(methylthio)-benzaldehyde (Scheme S1) to extend the π-conjugation and form a chromophore with push-pull character. The pentafluorophenyl served as an electron-withdrawing group and the thio-ether group acts as an electron-donating group to form a typical intermolecular charge transfer (ICT) dye. The electron-donating thio-ether group in this probe can be readily oxidized to electron-withdrawing SO group by HClO/ClO−, which leads to noticeable ratiometric florescence changes through a diminished ICT character (Scheme 1). The chemical structures of S-BODIPY, alongside the oxidized probe SO-BODIPY (Scheme S2), were fully characterized by HR-MS, 1H NMR and 13C NMR. The spectra along with detailed synthetic procedures can be found in the Supporting Information (Figure S7-S11). Insert Scheme 1

Spectral response of S-BODIPY to hypochlorite To test the feasibility of our design, we examined the sensing behaviour of S-BODIPY toward HClO/ClO− in PBS. As shown in Figure 1a and 1b, S-BODIPY (5 µM) displays a broad absorption band centred at 594 nm (λabs = 594 nm) and a fluorescence band centred at 619 nm with a moderate fluorescence quantum yield (Φfl = 0.147). Upon the addition of 100 equiv. of


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ClO−, the probe immediately displayed a dramatic hypsochromic fluorescence response. The time-course fluorescence spectra showed that the fluorescence ratio (F587/F619) promptly increased to a maximum within 30 seconds after the addition of ClO− and maintained this ratio for at least 3 min (Figure 1c). Insert Figure 1 Upon adding increasing ClO− to a solution of S-BODIPY, the absorption band centred at 594 nm decreased gradually and a new absorption band appeared at 578 nm with a well-defined isosbestic point at 582 nm (Figure 1a). The fluorescence emission band at 619 nm decreased and concomitantly a new emission band appeared at 587 nm (Figure 1b), providing a ratiometric manner for the detection of ClO−. Interestingly, the fluorescence intensity of S-BODIPY was enhanced about 7-fold after treatment with ClO−, which can be another alternative signal for imaging ClO− in living animals. This blue-shift in the absorption and fluorescence spectra is most likely a result of a reduction of the electron-donating ability of the thio-ether upon oxidation. To verify this hypothesis, we prepared a model compound, SO-BODIPY, in which the SCH3 group was substituted by the corresponding sulfoxide. As shown in Figure S1, the UV–Vis absorption and fluorescence spectra of S-BODIPY in the presence of ClO− were virtually identical to that of a pure sample of SO-BODIPY, clearly supporting the S-BODIPY to SO-BODIPY oxidation hypothesis. The fluorescent detection limit was calculated to be 59 nM (Figure S2), indicating good performance in comparison with some reported fluorescent probes for ClO− (Table S1). Thus, both the reaction kinetics and the probe’s sensitivity are ideally suited for the real-time imaging of small transient ClO− concentrations in living cells and organisms. Selectivity of S-BODIPY towards hypochlorite To evaluate the selectivity of S-BODIPY for ClO−, we examined the fluorescence response of the probe toward various ROS including reactive oxygen species, RNS, as well as other biologically relevant species. As shown in Figure 2a and Figure S3, the probe showed a negligible response to H2O2, •OH, TBHP, ROO• and •O2−. Furthermore, the probe did not exhibit any fluorescence response toward the RNS, and the reactive small biomolecules at biologically relevant concentrations. By contrast, an obvious colour change and fluorescence ratio



enhancement was observed after treatment of S-BODIPY with 0.5 mM ClO−. Additionally, the probe exhibits stable fluorescence output against pH changes under physiological conditions (Figure 2b). These results demonstrate that S-BODIPY has an excellent selectivity for ClO− over other biological species. Insert Figure 2 Spectroscopic analysis of the oxidation of S-BODIPY To investigate the sensing mechanism, S-BODIPY was treated with 28 equiv. NaClO in PBS for 10 min, and the major fluorescent product was separated by silica gel chromatography and the 1H

NMR and HR-MS were recorded. As shown in Figure S4, the chemical shift of SCH3 at 2.52

ppm was shifted downfield to 2.75 ppm after treatment with 28 equiv. NaClO, and the chemical shift of the adjacent protons on the phenyl ring at 7.25 ppm was shifted downfield to 7.65 ppm. Thus, the deshielding of the neighbouring nuclei clearly indicated an oxidation of the SCH3. HR-MS also confirmed the addition of an oxygen atom to S-BODIPY upon transformation to SO-BODIPY after treatment with ClO−. Hence, both 1H NMR and HR-MS results confirmed the oxidation pathway of the sulphur atom as depicted in Scheme 1. Cytotoxicity and photostability of S-BODIPY The cytotoxicity of S-BODIPY was evaluated using a CCK-8 assay. As shown in Figure S5, the cell survival rate after incubation with S-BODIPY remained above 77%, demonstrating an excellent biocompatibility with a clinically permissible dose of approximately 10 μM. As the photostability of imaging probes is one of the major hurdles to overcome in the design and development of imaging probes, we investigated the photostability of S-BODIPY (1 μM) in living cells under continuous irradiation with a confocal microscope. As shown in Figure S6, S-BODIPY demonstrated bright fluorescence in living cells, indicating a good cellular uptake of the dye. The fluorescence intensity of S-BODIPY inside living cells almost remained unchanged for 35 min, confirming a good photostability. The photostability of the probe after treatment with ClO− was also investigated (Figure S6-S7), showing a slight drop in the yellow fluorescence intensity within the first 10 minutes, but the fluorescence intensity remained more stable


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afterwards. The excellent photostabilities were ascribed to the non-reactive 5F-BODIPY parent structure, which can resist photo-oxidation and photo-bleaching. S-BODIPY could thus be suitable for the real-time monitoring of ClO− in vivo for an extended time. Ratiometric imaging of exogenous hypochlorite in living cells We then explored the potential of S-BODIPY for sensing cellular ClO−. Firstly, HeLa cells were incubated with S-BODIPY (1 μM) for 30 min at 37 °C, and then were subjected to confocal laser scanning microscopy. As shown in Figure 3a, the HeLa cells exhibited bright fluorescence in the red channel, but low intracellular fluorescence was detected from the yellow channel (552–617 nm). When the S-BODIPY-loaded HeLa cells were further treated with different concentrations of exogenous ClO− (5, 10 and 15 μM) for 10 min, the yellow fluorescence intensity increased dramatically, while the fluorescence in the red channel decreased markedly as well. Importantly, the probe exhibits a different ratiometric response toward various concentrations of hypochlorite with high sensitivity (Figure 3b and 3c). Insert Figure 3

Ratiometric imaging of exogenous and endogenously induced hypochlorite in zebrafish The excellent fluorescence properties of S-BODIPY prompted us to test its potential application for imaging HClO/ClO– in living zebrafish. We examined the background fluorescence of zebrafish in the absence of S-BODIPY and HClO/ClO–, as shown in Figure 5a, no luminescence was observed upon excitation at 543 nm. Then, zebrafish were incubated with S-BODIPY (1 μM) for 30 min. As shown in Figure 4a, a bright image was observed in the red channel, while only very weak yellow fluorescence was detected in the yellow channel, indicating that S-BODIPY can be easily absorbed by the zebrafish and distributed throughout the whole organism. As the concentrations of ClO– increased from 0 to 15 μM, the yellow fluorescence became much brighter (Figure 4b). The fluorescence ratio images displayed a clear colour evolution after the zebrafish were incubated with ClO− for 10 min, which provides a direct visualization of the ClO− level in vivo (Figure 4c). Interestingly, the ratiometric images showed that the level of ClO– in liver and intestine are significantly higher than other organs after



incubation with ClO– for 10 min. This phenomenon might be ascribed to the fundamental physiological functions of liver and intestine for the elimination of toxic species,41 confirming the possibility that these organs are primarily affected by high concentrations of HClO/ClO–.42 Insert Figure 4

To evaluate the feasibility of S-BODIPY for imaging endogenous ClO− in vivo, zebrafish were exposed to Lipopolysaccharide (LPS) (1 μM) in E3 embryo medium for 24 h at 28°C, and then the fluorescence images were recorded. As shown in Figure 5, a bright yellow fluorescence image was observed, which further confirmed the native HClO can be measured by S-BODIPY. From the ratiometric images, it can be observed that the LPS-induced HClO level in the liver was much higher than other organs, suggesting that endogenous ClO− was mainly generated in the liver after stimulation with LPS. Insert Figure 5

Ratiometric imaging of hypochlorite in a liver disease mouse model Lipopolysaccharide and D-galactosamine (LPS/DGalN) administration to mice has frequently been used as a model for endotoxemic shock and results in severe hepatotoxicity and associated reactive oxygen stress.43 In the current work, LPS/DGalN was administered to mice intraperitoneally, prior to S-BODIPY administration via tail vain injection, alongside a control group (Figure 6a). The induction of hepatic damage in the mice was confirmed by increased plasma levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) activity, common markers of hepatic injury and inflammation (Figure 6b). Furthermore, the very strong disease model-associated increases in tumour necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) in plasma levels of LPS/DGalN- treated mice confirmed the successful induction of liver damage in these mice (Figure 6d).44 As can be seen in Figure 6c, the induction of acute hepatic injury results in a clear ratiometric shift in the fluorescence of the yellow (550–610 nm) and red channels (610–670 nm) in vivo upon excitation at 550 nm. Thus, S-BODIPY is particularly well-suited for in vivo imaging of hypochlorite in living animals owing to the high sensitivity to hypochlorite, as well as long emission wavelengths and high fluorescence intensities.


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Insert Figure 6

Conclusions In conclusion, we have developed a novel BODIPY-based ratiometric fluorescent probe S-BODIPY for the rapid detection of ClO− to enable real time imaging of ClO− in vitro and vivo. The great merits of S-BODIPY are an excellent photostability, high fluorescence quantum yield and stable fluorescence output against pH changes. These features combined with its low cytotoxicity and good membrane permeability, enable the probe to serve as an ideal indicator for tracking ClO− in vitro and in vivo. Moreover, the probe was successfully applied to image the endogenous and exogenous HClO/ClO− in vivo in a ratiometric manner in zebrafish and mice.

Conflicts of interest There are no conflicts to declare.

Acknowledgements We gratefully thank the financial support from the National Natural Science Foundation of China (No.21203138, LZ) and the Natural Science Foundation of Tianjin (17JCYBJC19600, LZ). This work was also supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (CRI project no. 2018R1A3B1052702, JSK), and the Basic Science Research Program (2017R1D1A1B03032561, PV and 2017R1D1A1B03030062, MW) funded by the Ministry of Education as well as the Korea Research Fellowship Program funded by the Ministry of Science and ICT through the National Research Foundation of Korea (2016H1D3A1938052, PV).

Supporting Information Synthesis of SO-BODIPY, absorption and fluorescence spectra, cytotoxicity, photostability, 1H and 13 C NMR spectra.



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Figures and Captions F

F F

F

F

F

F

F

F

F

ClO ─

N N B F F

N N B F F

S

O

S

Scheme 1 Sensing mechanism of hypochlorite by S-BODIPY

Fl intensity (a.u.)

0 μM

0.6

[ClO-] 480 μM

0.3 0.0

(c)

(b)

0.9

500 550 600 Wavelength (nm)

4000 [ClO-]

2000

(d)

0 550

4 2

600 650 Wavelength (nm)

700

1.0 0.8

6

0 μM

1000

650

8

480 μM

3000

F587 / F619

Absorbance

(a)

F587 / F619

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

y = 0.0060x + 0.1073 R2 = 0.9911

0.4 0.2

0 0

80 160 Time (s)

240

0

40 80 [ClO-] (μM)

120

Figure 1. (a) UV-vis absorption spectra and (b) fluorescence spectral changes of S-BODIPY (5 μM) in DMF/PBS solution (v/v = 1/1, pH 7.4, 10 mM) upon addition of increasing amounts of NaClO (0 – 480 μM). Each spectrum was recorded after 1 min. (c) Time dependent fluorescence emission ratios (F587/F619) response of S-BODIPY (5 μM) to NaClO (0.5 mM) in DMF/PBS solution (v/v = 1/1, pH 7.4, 10 mM). (d) Linear relationships between fluorescence intensity ratios (F587/F619) of S-BODIPY (5 μM) versus concentrations of NaClO. The excitation wavelength was 540 nm. Slits: 2.5/2.5 nm.



(b) 8

6

6

4 a b c de f g h i j k l mno p q r s t u v w x

F587 / F619

(a) 8

F587 / F619

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2 0

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4 2

S-BODIPY S-BODIPY + ClO

0 abcdefghijklmnopqrstuvwx

4

5

6

7

pH

8

9

10

Figure 2. (a) Fluorescence emission ratios (F587/F619) of S-BODIPY (5 μM) in DMF/PBS solution (v/v = 1/1, pH 7.4, 10 mM) in the presence of various small molecular species (0.5 mM) and ROS/RNS (100 μM). a, only the probe; b, HS−; c, HSO3−; d, Cl−; e, HPO42−; f, AcO−; g, NO2−; h, NO3−; i, H2PO4−; j, H2O2; k, GSH; l, Hcy; m, Cys; n, S2O32−; o, SO32−; p, HCO3−; q, PO43−; r, •OH; s, TBHP; t, TBO•; u, •O2−; v, NO; w, ONOO−; x, ClO−. Insert: fluorescence images of S-BODIPY (5 μM) in the presence of various small reactive species at relavent concentrations. (b) pH dependent fluorescence emission ratios (F587/F619) response of S-BODIPY (5 μM) to NaClO (0.5 mM) in DMF/PBS solution (v/v = 1/1, 10 mM). Each spectrum was recorded after 1 min. The excitation wavelength was 540 nm. Slits: 2.5/2.5 nm.

Figure 3. (a) Confocal ratiometric fluorescence images (λex = 543 nm, yellow channel (λem = 552– 617 nm) and red channel (λem = 648–708 nm)) of S-BODIPY in HeLa cells incubated with different concentrations of ClO−. HeLa cells were incubated with S-BODIPY (1 μM) at 37 °C for 30 min, and then further treated with different concentrations of ClO− for 10 min. (b) Fluorescence intensities were measured as averages of 5 regions of interest (ROIs) from different treated HeLa cells. (c) Fluorescence intensity ratios were measured as averages of 5 regions of interest (ROIs) from different treated HeLa cells. Scale bar: 20 μm.


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Figure 4. (a) Ratiometric fluorescence images (λex = 543 nm, yellow channel (λem = 552–617 nm) and red channel (λem = 648–708 nm)) of of four-day old zebrafish loaded with S-BODIPY (1 μM) for 0.5 h, followed by incubation with different amount of ClO− for 10 min at 28°C. (b) Fluorescence intensities were measured as averages of 5 regions of interest (ROIs) in zebrafish. (c) Fluorescence intensity ratios were measured as averages of 5 regions of interest (ROIs) in zebrafish. Scale bar: 500 μm. The local ratio scale bar: 100 μm.



Figure 5. (a) Fluorescence imaging of endogenous HClO production in zebrafish with S-BODIPY (1 μM) during an LPS-mediated inflammatory response. Ratiometric images (λex = 543 nm, yellow channel (λem = 552–617 nm) and red channel (λem = 648–708 nm)) of four-day old zebrafish loaded with the S-BODIPY (1 μM) for 0.5 h and the fluorescence images of S-BODIPY−loaded zebrafish after treated with LPS for 24h at 28 °C. (b) Fluorescence intensities were measured as averages of 5 regions of interest (ROIs) in zebrafish. (c) Fluorescence intensity ratios were measured as averages of 5 regions of interest (ROIs) in zebrafish. Scale bar = 500 μm. The local ratio scale bar: 100 μm.


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Figure 6. In vivo imaging of endogenous HClO in an acute liver injury mouse model. (a) Mice injected with control (PBS only) and S-BODIPY (1 mM, 30% EtOH, 30%, DMF) by i.v. injection pretreated with LPS (10 μg/kg)/D-GalN (700 mg/kg)-injected mice for 12 h (group: n=5). (b) Plasma AST and ALT activity in control and LSP/D-GalN treated mice for 12 h. (c) In vivo imaging of endogenous HClO production from the peritoneal cavity of the mice treated with LPS/D-GalN. Representative images of mice intravenously treated with S-BODIPY (1 mM) for 2 h, pre-treated via intraperitoneal injection of PBS (left) and LPS/D-GalN (right). Ratiometric fluorescence images of mice (λex = 550 nm, yellow channel (λem = 550–610 nm) and red channel (λem = 610–670 nm)). (d) Effects of LPS/D-GalN on proinflammatory cytokines.



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In Vivo Imaging of Endogenously Produced HClO in Zebrafish and

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